Tyrosinase Multilayer-Functionalised Carbon Nanotubes as Electrochemical Labels: Application To Immunoassay Porramate Chumyim & Patsamon Rijiravanich & Mithran Somasundrum & Werasak Surareungchai Published online: 11 July 2014 # Springer Science+Business Media New York 2014 Abstract We have modified carboxylic acid-functionalised multi-walled carbon nanotubes (MWNTs) with multilayers of tyrosinase/polyallylamine using a layer-by-layer process. Based on optical determination, each tyrosinase deposition coated approximately three layers of enzyme onto the nano- tubes. Based on kinetic analysis of the steady-state current of the modified MWNTs to catechol, the minimum value of k cat / K M for the immobilised enzyme was 3.9×10 8 cm 3 s −1 mol −1 ± 0.5×10 8 cm 3 s −1 mol −1 (n =3), which is one order of magni- tude less than the value for the enzyme in solution. As an illustration of an immunoassay application, the modified MWNTs were coated with streptavidin and then used as electrochemical labels for biotin-tagged anti-rabbit Salmonella polyclonal antibodies. Following optimisation of enzyme layer number, pH, incubation time and electrochem- ical parameters, heat-killed Salmonella Typhimurium cells were calibrated across the range 1 CFU ml − 1 to 10 7 CFU ml −1 by direct enzyme-linked immunosorbent assay (ELISA) with a limit of detection (signal/noise=3) of approx. 340 CFU ml −1 . Keywords Layer-by-layer . Tyrosinase . Carbon nanotube . Salmonella . Immunoassay 1 Introduction Redox cycling is the regeneration of an electro-active species in the vicinity of an electrode. The regeneration can be chem- ical or electrochemical, and will result in an amplified Faradaic signal as the signaling species is cycled between oxidised and reduced forms. This suggests the possibility of sensitive electrochemical measurement. In the case of electro- chemical cycling, the analyte itself is cycled between closely spaced microelectrodes [1]. In chemical cycling, a species reacts with a mediator or an enzyme. Since the mediator or enzyme can be attached to a label, this form of cycling can be used in biosensor construction to detect a biological binding event, such as antibody to antigen or DNA hybridisation [2]. In general, electrochemical biosensors have the advantage of producing an electrical signal without needing expensive transduction equipment, and have the possibility of being miniaturised. The advantage of mediator cycling is that it can be expected to be relatively stable, provided the mediator is retained on the support. The advantage of an enzyme is that the rate of the enzyme-substrate reaction is usually much faster. The response of an enzyme label can be amplified by using a second enzyme to regenerate a product of the first enzyme reaction [3]. To achieve a good level of amplification, the second enzyme should have a good rate of reaction for this species and should be present at a high concentration [4]. Whether it is possible to achieve this will depend on the inherent catalytic rate (k cat ) of the enzyme for the substrate, Electronic supplementary material The online version of this article (doi:10.1007/s12668-014-0144-7) contains supplementary material, which is available to authorized users. P. Chumyim : W. Surareungchai School of Bioresources and Technology, King Mongkut’ s University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand P. Rijiravanich : M. Somasundrum (*) Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and Biotechnology, National Sciences and Technology Development Agency, King Mongkut’ s University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand e-mail: mithran.somasundrum@gmail.com W. Surareungchai (*) Biological Engineering Graduate Program, King Mongkut’ s University of Technology Thonburi, Bangmod, Bangkok 10140, Thailand e-mail: werasak.sur@kmutt.ac.th BioNanoSci. (2014) 4:240–250 DOI 10.1007/s12668-014-0144-7